Potential impact of efflux pump genes in mediating rifampicin resistance in clinical isolates of Mycobacterium tuberculosis from India.

Anshika Narang,Mandira Varma-Basil,Mridula Bose,Kamal Shrivastava,Kushal Garima,Shraddha Porwal,Astha Giri,Archana Bhandekar,Naresh Kumar Sharma,Shampa Anupurba

doi:10.1371/journal.pone.0223163

Anshika Narang, Mandira Varma-Basil + Show 8 more

Open Access

https://doi.org/10.1371/journal.pone.0223163

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Journal: PloS one	Publication Date: Sep 26, 2019
Citations: 13	License type: CC BY 4.0

Affiliation: University of Delhi

Abstract

Despite the consideration of chromosomal mutations as the major cause of rifampicin (RIF) resistance in M. tuberculosis, the role of other mechanisms such as efflux pumps cannot be ruled out. We evaluated the role of four efflux pumps viz., MmpL2 (Rv0507), MmpL5 (Rv0676c), Rv0194 and Rv1250 in providing RIF resistance in M. tuberculosis. The real time expression of the efflux pumps was analyzed in 16 RIF resistant and 11 RIF susceptible clinical isolates of M. tuberculosis after exposure to RIF. Expression of efflux pumps in these isolates was also correlated with mutations in the rpoB gene and MICs of RIF in the presence and absence of efflux pump inhibitors. Under RIF stress, Rv0194 was induced in 8/16 (50%) RIF resistant and 2/11 (18%) RIF susceptible isolates; mmpL5 in 7/16 (44%) RIF resistant and 1/11 (9%) RIF susceptible isolates; Rv1250 in 4/16 (25%) RIF resistant and 2/11 (18%) RIF susceptible isolates; and mmpL2 was upregulated in 2/16 (12.5%) RIF resistant and 1/11 (9%) RIF susceptible isolates. This preliminary study did not find any association between Rv0194, MmpL2, MmpL5 and Rv1250 and RIF resistance. However, the overexpression of Rv0194 and mmpL5 in greater number of RIF resistant isolates as compared to RIF susceptible isolates and expression of Rv0194 in wild type (WT) resistant isolates suggests a need for further investigations.

Highlights

We have explored the role of efflux pumps MmpL5, Rv1250, MmpL2 and Rv0194 in resistance to RIF by studying the expression of the genes encoding these membrane transporters under RIF stress, in M. tuberculosis isolates obtained from patients of pulmonary tuberculosis
Of the 130 isolates of M. tuberculosis tested by proportion method of drug susceptibility testing (PDST), 33 were found to be RIF resistant and 97 were RIF susceptible
Presence or absence of mutations in the rpoB RIF resistance-determining region (RRDR) was confirmed by sequencing in 30 RIF resistant and 11 RIF susceptible isolates

Summary

Methods

Clinical isolates (n = 130) of M. tuberculosis and the reference strain H37Rv were collected from the Department of Microbiology at Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India. The clinical isolates were obtained as part of a convenience sample obtained from new smear positive patients of pulmonary tuberculosis attending the Department of Respiratory Medicine at Vallabhbhai Patel Chest Institute which serves as a referral center for patients with respiratory diseases in North India. Cultures were grown on Lowenstein Jensen (LJ) medium slants and in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) supplemented with OADC (oleic acid, albumin bovine, fraction V, dextrose, catalase) (Difco) and 0.2% glycerol at 37 ̊C. All the isolates (n = 130) were characterized by niacin, nitrate and catalase tests [14] and confirmed as M. tuberculosis complex (MTBC) by PCR Restriction Analysis (PRA) [15]

Results

Discussion

Conclusion